This renewal is a continuation of studies examining the molecular mechanisms of tachykinin peptide and receptor synthesis, function and regulation. Members of this peptide family are neurotransmitter candidates in many neuronal systems, and consist of substance P (SP), neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK) and neuropeptide gamma (NPgamma), which act at three characterized G-protein coupled receptors, called the SP receptor (SPR), NKAR and NKBR. We have characterized mechanisms of SP biosynthesis and regulation by examining the structure of the SP/NKA gene, the three mRNAs produced via differential RNA splicing, secretory pathway targeting of the translated precursors, and precursor processing mechanisms. We have also characterized the SPR receptor cDNA and gene by molecular cloning and functional expression. In this proposal, we shall pursue four specific aims to examine peptide receptor responses, to investigate cellular and molecular mechanisms regulating receptor sensitivity, and to evaluate regulation of peptide synthesis and receptor responsiveness in a physiological system. 1) Structure-activity relationships of the rat SPR will be determined by examining high affinity agonist binding of chimeric, deletion and site-directed mutant SPR forms. 2) The synthesis, assembly and turnover of the SPR will be examined in native SPR bearing cell lines in pulse-chase experiments using site-directed anti-SPR antisera, and the effects of cellular activation by second messages on these processes will be further assessed. 3) Agonist-dependent second messenger systems regulating SPR gene expression and mRNA turnover in native SPR bearing cell lines will be investigated to explore the hypothesis that acute and chronic SPR regulation involve transcriptional mechanisms. 4) SP synthesis, turnover and receptor functions will be studied in spinal sensory systems in vivo. Spinal sensory systems related to nociception will be activated using chemogenic or other pain models (formalin test, adjuvant induced polyarthritis, partial sciatic nerve ligation) and function, turnover and regulation of tachykinin peptides and receptors will be assessed. These studies will provide a basis for understanding the molecular actions of tachykinins at target sites and their regulation. They also provide a genetic basis for investigating whether aberrations in the biosynthesis of SP and its receptor are responsible for or associated with sensory and neurodegenerative disorders in which CNS peptides are implicated.